FTO O-GlcNAcylation promotes TRIM21-mediated FTO ubiquitination degradation to sustain the negative feedback control of macrophage inflammation

Introduction

The intricate interplay of molecular pathways constitutes the foundational machinery governing a vast array of physiological processes, all of which are absolutely essential for maintaining cellular homeostasis, enabling precise responses to various environmental cues, and mounting a robust defense against invading pathogens. Within this complex and highly regulated biological network, the protein known as fat mass and obesity-associated protein, or FTO, has increasingly garnered substantial scientific attention due to its multifaceted roles. FTO functions as a pivotal RNA N6-methyladenosine (m6A) demethylase, an enzymatic role that designates it as a key player in the dynamic landscape of epitranscriptomics. As a demethylase, FTO is specifically responsible for the removal of a crucial chemical modification, the N6-methyladenosine mark, from messenger RNA (mRNA) molecules. This m6A modification itself represents a widespread epigenetic mark that profoundly influences various aspects of gene expression, thereby exerting control over processes such as the stability of mRNA molecules, their alternative splicing patterns, their efficiency of translation into proteins, and their precise cellular localization within the cell.

In recent years, an accumulating body of research has progressively highlighted FTO’s significant and multifaceted involvement in the complex orchestration of the inflammatory response. Inflammation, while an undeniably vital biological process for host defense against infection and injury, can paradoxically contribute to a wide range of pathological conditions when its delicate regulatory balance is disrupted or when it becomes dysregulated and prolonged. Concurrently, an emerging and increasingly compelling body of evidence has begun to firmly establish a strong conceptual link between O-GlcNAcylation, a unique and highly dynamic post-translational modification. This modification involves the rapid and reversible attachment of a single N-acetylglucosamine sugar molecule to the serine and threonine residues of proteins, influencing their structure, function, and interactions. O-GlcNAcylation has been implicated in numerous human diseases, with a particularly strong and recurrent association observed in various inflammatory conditions. Despite the individual recognition of FTO’s established role in inflammatory processes and the growing understanding of O-GlcNAcylation’s pervasive influence on a multitude of cellular functions, the precise role and the specific, intricate underlying molecular mechanisms through which FTO O-GlcNAcylation participates in the complex regulation of inflammation have remained largely elusive. This significant knowledge gap represents a critical frontier in our understanding of inflammatory disease. Therefore, this study was systematically undertaken with the explicit aim to meticulously delineate these intricate molecular connections and, in doing so, to uncover a novel and therapeutically pertinent regulatory axis directly involved in inflammatory processes, potentially paving the way for new therapeutic interventions.

Methods

To meticulously unravel the complex and previously undefined molecular mechanisms that govern FTO’s role in inflammation, and more specifically, to precisely ascertain the nature and functional impact of its O-GlcNAcylation, a comprehensive and cutting-edge suite of advanced experimental methodologies was rigorously employed. The direct identification and precise localization of the O-GlcNAcylation modification on the FTO protein itself were determined through a multi-pronged approach involving highly sensitive and specific techniques. Initially, co-immunoprecipitation assays were utilized to detect direct physical interactions between proteins, or modifications thereof, by selectively precipitating a protein complex using an antibody. This approach was complemented by metabolic glycan labeling, a technique that involves the incorporation of a chemically modified sugar into cellular proteins, which allowed for the specific detection and subsequent isolation of O-GlcNAcylated proteins using a bioorthogonal click reaction. The precise mapping of these modifications, providing definitive identification of the specific amino acid residues that were O-GlcNAcylated, was then achieved through detailed liquid chromatography-tandem mass spectrometry analysis, offering unparalleled molecular resolution.

To thoroughly investigate the upstream regulatory elements controlling FTO O-GlcNAcylation, particularly focusing on the expression of glutamine-fructose-6-phosphate amidotransferase 2 (GFAT2), an enzyme of paramount importance within the hexosamine biosynthetic pathway that generates UDP-GlcNAc, the crucial substrate for O-GlcNAcylation, chromatin immunoprecipitation coupled with quantitative polymerase chain reaction (ChIP-qPCR) was extensively utilized. This powerful technique enabled the precise determination of the binding of the transcription factor FOXO1 to the promoter region of the Gfat2 gene, thereby directly assessing its transcriptional regulatory role. Concurrently, to measure the direct transcriptional activity of the Gfat2 promoter under inflammatory stress, dual-luciferase reporter assays were meticulously conducted during lipopolysaccharide (LPS) stimulation. These assays provided invaluable insights into the dynamic transcriptional responses that govern GFAT2 expression under acute inflammatory conditions.

The investigation extended to the critical process of FTO protein stability, specifically examining its ubiquitination modification and its physical interaction with the E3 ubiquitin ligase TRIM21. Confocal microscopy was employed to visually discern the cellular localization of FTO and TRIM21, and to observe their potential colocalization, which would be indicative of a direct physical association within the cellular compartments. Direct protein-protein interactions were then unequivocally confirmed through rigorous pull-down assays, providing biochemical evidence of their physical association. Furthermore, the specific ubiquitination sites on FTO and the precise nature of the ubiquitin chains attached to it were meticulously elucidated through detailed mass spectrometry analysis, thereby providing intricate molecular details regarding this crucial post-translational modification. To directly assess the functional consequences of FTO O-GlcNAcylation on its ubiquitination-mediated degradation, additional experiments were performed. Co-immunoprecipitation was again employed to detect any altered ubiquitination patterns in response to varying O-GlcNAcylation statuses of FTO. Crucially, protein stability assays were conducted to directly measure the half-life of FTO variants, comparing those with and without the O-GlcNAcylation modification, thereby providing empirical evidence for its direct impact on FTO protein turnover and stability.

Moreover, to gain a comprehensive understanding of the downstream molecular effects resulting from FTO degradation, the levels of m6A methylation on the messenger RNA of suppressor of cytokine signaling 1 (Socs1) were precisely detected. This was achieved using the highly specific technique of m6A-RNA-immunoprecipitation (RIP)-qPCR. This methodology allowed for the specific enrichment of m6A-modified RNA fragments, followed by their accurate quantification, thus providing direct evidence of altered m6A methylation patterns on Socs1 mRNA in response to changes in FTO levels.

Finally, to bridge these molecular and cellular findings with broader physiological contexts and to rigorously evaluate systemic inflammatory responses in a living organism, a series of crucial in vivo experiments were conducted using established mouse models of sepsis. Myeloid-specific Fto deletion (myeFto -/-) mice were specifically generated to selectively ablate FTO within myeloid cells, a lineage that includes macrophages, thereby allowing for a targeted assessment of FTO’s indispensable role in innate immunity. To further confirm the cell-specific effects of FTO, macrophage depletion and subsequent reconstitution experiments were meticulously performed. Inflammatory responses in these sophisticated mouse models were rigorously evaluated following the induction of sepsis, utilizing either systemic administration of *Salmonella Typhimurium* infection, a bacterial pathogen that elicits a robust immune response, or bacterial endotoxin (lipopolysaccharide, LPS), a potent mimic of Gram-negative bacterial infection. Systemic levels of crucial inflammatory cytokines within the mouse models were quantitatively assessed using highly sensitive enzyme-linked immunosorbent assays (ELISA), which provided a direct and precise measure of the overall systemic inflammatory state. Collectively, this comprehensive and multi-modal array of experimental approaches provided a robust and irrefutable framework for dissecting the intricate and previously undefined role of FTO O-GlcNAcylation in the nuanced regulation of inflammatory signaling, culminating in a profound new understanding.

Results

Our extensive and meticulous investigation yielded several novel and highly significant findings regarding the intricate regulation and dynamic function of FTO within the complex landscape of inflammatory responses. A foundational and critical discovery was the precise identification that the FTO protein undergoes a specific O-GlcNAcylation modification. This post-translational addition of a sugar molecule was found to be remarkably site-specific, occurring exclusively at the serine 95 (Ser95) residue located within the FTO protein sequence. This precise molecular detail provides an essential structural and mechanistic basis for comprehending the exact regulatory role played by this particular post-translational modification.

Further in-depth probing into the upstream molecular mechanisms governing the O-GlcNAcylation of FTO revealed a direct and profound link to inflammatory stimuli. We demonstrated conclusively that stimulation with lipopolysaccharide (LPS), a potent bacterial endotoxin frequently employed in experimental models to mimic systemic inflammation, significantly and robustly enhances the overall levels of FTO O-GlcNAcylation modification. This observed increase was not a random occurrence but was mechanistically driven through the transcriptional upregulation of glutamine-fructose-6-phosphate amidotransferase 2 (GFAT2) expression. Critically, the expression of GFAT2 itself was found to be under the direct transcriptional control of the FOXO1 transcription factor. Thus, the LPS stimulus initiates a cascade: it triggers an increase in FOXO1 activity, which consequently leads to elevated GFAT2 levels. This increase in GFAT2, in turn, boosts the cellular supply of UDP-GlcNAc, the essential substrate required for O-GlcNAcylation, ultimately culminating in a marked enhancement of FTO O-GlcNAcylation.

Perhaps the most pivotal and functionally impactful discovery was the comprehensive elucidation of the direct consequence of FTO O-GlcNAcylation. We unequivocally found that the O-GlcNAcylation of FTO specifically and robustly promotes its ubiquitination-mediated degradation. This intricate degradation pathway is precisely orchestrated by the E3 ubiquitin ligase TRIM21, a key enzyme in the ubiquitin-proteasome system. This process involves the formation of K48-linked ubiquitin chains on the FTO protein, a specific type of ubiquitin linkage that is well-established to target proteins for rapid and efficient degradation by the proteasome. The accelerated degradation of FTO, which is exquisitely orchestrated by its O-GlcNAcylation status, subsequently exerts profound and far-reaching downstream effects on gene expression, particularly on genes highly relevant to inflammatory processes. Specifically, the reduced cellular levels of FTO, consequent to its degradation, lead to a direct increase in N6-methyladenosine (m6A) methylation on the messenger RNA (mRNA) of suppressor of cytokine signaling 1 (Socs1). Elevated m6A levels on Socs1 mRNA have the functional consequence of enhancing the stability of the mRNA and sustaining the expression of the SOCS1 protein. SOCS1 is a well-characterized and crucial negative feedback regulator of cytokine signaling, primarily exerting its inhibitory effects by blocking the activity of the JAK-STAT pathway, a central signaling cascade in inflammation. Consequently, the sustained SOCS1 protein expression acts to effectively suppress the excessive production of multiple key pro-inflammatory cytokines, including interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), specifically within macrophages that have been stimulated by LPS. This intricate and precisely regulated molecular cascade highlights a novel and essential regulatory loop where FTO O-GlcNAcylation ultimately serves to dampen and control the inflammatory response.

The profound physiological relevance and therapeutic potential of this newly identified regulatory mechanism were robustly and compellingly validated through a series of rigorous in vivo studies utilizing established murine models of inflammation and sepsis. Introducing a specific FTO O-GlcNAcylation mutation (S95A), which renders the FTO protein incapable of being O-GlcNAcylated at the critical Ser95 site, resulted in a significant and measurable aggravation of inflammatory responses in mouse models of sepsis induced by either *Salmonella Typhimurium* infection or systemic LPS administration. This direct experimental evidence unequivocally demonstrated that the absence of FTO O-GlcNAcylation exacerbates the septic phenotype, leading to a more severe inflammatory reaction. Conversely, in wild-type mice, where physiological O-GlcNAcylation of FTO can occur naturally, this modification was consistently shown to suppress the hyperinflammatory phenotype, effectively acting as a natural and intrinsic brake on excessive inflammation, thereby protecting the host. Further supporting the potential therapeutic implications of this pathway, pharmacological promotion of overall protein O-GlcNAcylation using Thiamet-G, a widely recognized inhibitor of O-GlcNAcase (OGA, the enzyme responsible for removing O-GlcNAc from proteins), successfully alleviated LPS-induced inflammatory responses and significantly mitigated the severity of septic shock in mice. This comprehensive and consistent set of results, spanning molecular, cellular, and organismal levels, collectively establishes a critical and previously unrecognized role for FTO O-GlcNAcylation in dynamically modulating inflammatory responses both in controlled in vitro settings and complex in vivo physiological environments.

Conclusion

These seminal findings collectively unveil a previously unrecognized, yet critically important, regulatory mechanism wherein the O-GlcNAcylation of the fat mass and obesity-associated protein, FTO, assumes a pivotal and controlling role in modulating inflammatory responses. Our comprehensive study definitively demonstrates that this specific O-GlcNAcylation event on FTO directly promotes its ubiquitination-mediated degradation, a meticulously controlled cellular process executed by the E3 ubiquitin ligase TRIM21 and precisely involving the formation of K48-linked ubiquitin chains, a well-known signal for proteasomal destruction. This targeted degradation of FTO subsequently triggers a crucial and far-reaching downstream effect: it induces a significant increase in N6-methyladenosine (m6A) methylation levels on the messenger RNA (mRNA) of suppressor of cytokine signaling 1 (Socs1). The resulting heightened m6A methylation on Socs1 mRNA, in turn, acts to stabilize and sustain the cellular expression of the corresponding SOCS1 protein, which is itself a well-established and essential negative feedback regulator within inflammatory signaling pathways. Ultimately, this intricate molecular cascade effectively downregulates the inflammatory response initiated and mediated by lipopolysaccharide, particularly by suppressing the excessive production of key pro-inflammatory cytokines such as interleukin-1 beta (IL-1β), MK-8719 interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) in activated macrophages. This newly identified and complex regulatory loop is indispensable for maintaining the delicate balance of the immune system, serving as a critical negative feedback control mechanism that actively prevents an uncontrolled and potentially damaging macrophage-driven inflammatory cytokine storm, a pathological hallmark of severe inflammatory conditions like sepsis. The identification of this novel regulatory axis involving FTO O-GlcNAcylation not only significantly deepens our fundamental understanding of the complex molecular underpinnings of inflammation but also carries profound and promising implications for the future development of therapeutic strategies. The precise pharmacological or genetic regulation of FTO O-GlcNAcylation, either through direct modulation of the glycosylation process itself or by specifically targeting the enzymes involved in its addition or removal, may offer a highly promising and potentially potent therapeutic strategy for combating endotoxin-induced inflammatory diseases, such as the life-threatening conditions of sepsis and septic shock. Furthermore, this newly elucidated mechanism could be broadly relevant to a wider spectrum of pathological conditions characterized by abnormal expression or dysregulation of FTO, potentially opening novel avenues for treatment in various inflammatory disorders and beyond, thereby offering new hope for patients. This research, therefore, lays a vital and expansive foundation for future investigations into O-GlcNAcylation as a compelling and druggable target in the complex pathology of inflammation.